Method of electrochemically producing hydrogel, method of producing hydrogel with pattern formed of cells, hydrogel production apparatus, and transducer

ABSTRACT

A hydrogel is formed by a reaction which is induced, in an electrolytic solution, by an electrode product electrochemically generated by electrodes installed in the electrolytic solution. An apparatus including an electrolytic tank with a bottom surface on which a two-dimensional array of working electrodes is provided and a counter electrode installed in the electrolytic tank is prepared. An electrolytic solution containing a dissolved substance that causes electrolytic deposition of a hydrogel is housed in the electrolytic tank. By applying a predetermined voltage to one or more selected working electrodes of the two-dimensional array, a hydrogel with a two-dimensional pattern corresponding to the arrangement of the selected working electrodes is formed.

TECHNICAL FIELD

The present invention relates to a method of electrochemically producinga hydrogel by using electrolytic deposition, a method of producing ahydrogel with a pattern formed of cells, a hydrogel productionapparatus, and a transducer.

BACKGROUND ART

A hydrogel is used as a scaffold for cell culture, for example. A methodof electrochemically producing a hydrogel by using electrolyticdeposition is known.

Non-patent Literature 1 and Non-patent Literature 2 disclose methods ofelectrolytically depositing, on an indium tin oxide (ITO) electrode witha pattern, a hydrogel corresponding to the pattern. Non-patentLiterature 3 and Non-patent Literature 4 disclose methods ofelectrolytically depositing a hydrogel by using a Pt wire electrode orby making a Pt wire electrode perform scanning in an electrolyticsolution. Non-patent Literature 5 and Non-patent Literature 6 disclosetechniques of electrolytically depositing a hydrogel or the like, byapplying a voltage to one electrode selected from two electrodes or fromamong one-dimensional array of electrodes or, on the electrode.

PRIOR ART LITERATURE Non-Patent Literature

-   Non-patent Literature 1: F. Ozawa et al., “Cell Sheet Fabrication    Using RGD Peptide-Coupled Alginate Hydrogels Fabricated by an    Electrodeposition Method”, Chemistry Letters, Vol. 46, No. 4, pp    605-608, Advance Publication on the web Feb. 10, 2017.    <http://www.journal.csj.jp/doi/pdf/10.1246/c1.170003>-   Non-patent Literature 2: F. Ozawa et al., “Alginate gel microwell    arrays using electrodepotion for three-dimensional cell culture”,    Lab on a Chip, 2013, 13(15), pp 3128-3135.    <http://pubs.rsc.org/en/Content/ArticleLanding/2013/LC/c31c50455g#!div    Abstract>-   Non-patent Literature 3: F. Ozawa et al., “Electrochemical Hydrogel    Lithography of Calcium-Alginate Hydrogels for Cell Culture”,    Materials, 2016, 9(9), 744. <http://www.mdpi.com/1996-1944/9/9/744>-   Non-patent Literature 4: F. Ozawa et al., “Electrodeposition of    alginate gels for construction of vascular-like structures”, Journal    of Bioscience and Bioengineering, 2013, VOL 115, No. 4, pp 459-461.    <http://www.sciencedirect.com/science/article/pii/S1389172312004409?via    %3Dihub>-   Non-patent Literature 5: K. M. Gray et al., “Electrodeposition of a    Biopolymeric Hydrogel: Potential for One-Step Protein    Electroaddressing”, Biomacromolecules, 2012, 13(4), pp 1181-1189.    <http://pubs.acs.org/doi/abs/10.1021/bm3001155>-   Non-patent Literature 6: H. Yi et al., “Signal-Directed sequential    Assembly of Biomolecules on Patterned surfaces”, Langmuir, 2005,    21(6), pp 2104-2107. <http://pubs.acs.org/doi/abs/10.1021/1a047529k>

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

Non-patent Literatures 1 to 6 disclose the methods of electrolyticallydepositing a hydrogel. However, with the methods disclosed in Non-patentLiteratures 1 and 2, only two-dimensional patterning can be performed.In addition, since the methods disclosed in Non-patent Literatures 1 and2 make it necessary to produce an electrode for each pattern, themethods lack versatility. The methods disclosed in Non-patentLiteratures 3 and 4 are methods by which a pattern is formed by usingwire scanning, which is an extremely difficult and inefficient means,and can only form a limited and specific three-dimensional shape such asa tube. The techniques disclosed in Non-patent Literatures 5 and 6 aretechniques of forming a hydrogel on one selected electrode and do notaim to perform shaping.

The present invention provides a technique of electrochemicallyproducing a hydrogel, the technique that has a higher degree offlexibility in shaping, has greater versatility, and can form a hydrogelwith a two-dimensional pattern or a three-dimensional pattern moreefficiently than ever before.

Means to Solve the Problems

According to an invention of Claim 1, a method of electrochemicallyproducing a hydrogel by using electrolytic deposition of a hydrogel, themethod by which a hydrogel is formed by a reaction which is induced inan electrolytic solution by an electrode product electrochemicallygenerated by electrodes installed in the electrolytic solution, includesa step of preparing an apparatus including an electrolytic tank with abottom surface on which a two-dimensional array of working electrodes isprovided and a counter electrode which is installed in the electrolytictank, housing an electrolytic solution containing a dissolved substance,which causes electrolytic deposition of the hydrogel, in theelectrolytic tank, and, by applying a predetermined voltage to one ormore selected working electrodes of the two-dimensional array of theworking electrodes, forming, on the bottom surface, the hydrogel with atwo-dimensionally defined pattern corresponding to the arrangement ofthe selected working electrodes.

In an invention of Claim 2, the invention of Claim 1 includes: a firststep of forming a hydrogel with a two-dimensionally defined firstpattern by applying a first voltage as the predetermined voltage to afirst working electrode group as the one or more selected workingelectrodes; and a second step of forming a hydrogel with atwo-dimensionally defined second pattern by applying a second voltage asthe predetermined voltage to a second working electrode group as the oneor more selected working electrodes. The second step is performed afterthe first step, and a hydrogel with a three-dimensionally definedpattern including at least two layers formed of the first pattern andthe second pattern, which is located under the first pattern on the sidethereof closer to the bottom surface, is formed.

In an invention of Claim 3, in the invention of Claim 2, at least one ofthe first step and the second step includes a first sub-step and asecond sub-step, a corresponding one of the first working electrodegroup and the second working electrode group includes a first workingelectrode sub-group and a second working electrode sub-group, acorresponding one of the first voltage and the second voltage includes afirst sub-voltage and a second sub-voltage, and a corresponding one ofthe first pattern and the second pattern includes a first sub-patternand a second sub-pattern. The second working electrode sub-groupconsists of only the working electrode which does not belong to thefirst working electrode sub-group. A hydrogel with the first sub-patternis formed by applying the first sub-voltage to the first workingelectrode sub-group and a hydrogel with the second sub-pattern is formedby applying the second sub-voltage to the second working electrodesub-group.

In an invention of Claim 4, in the invention of Claim 3, in the secondsub-step, a hydrogel which dissolves under a predetermined condition isformed. The invention of Claim 3 further includes, at least after thefirst sub-step and the second sub-step, a sub-pattern removal step ofselectively removing the hydrogel with the second sub-pattern bydissolving the hydrogel with the second sub-pattern by providing thehydrogel with the second sub-pattern with the predetermined condition.

In an invention of Claim 5, in the invention of any one of Claims 2 to4, a Z stage that has an adhesion surface facing the bottom surfaceparallel thereto and is displaceable in a Z direction perpendicular tothe bottom surface is further prepared. By performing the first stepwith the Z stage being located in the electrolytic solution such thatthe adhesion surface is apart from the bottom surface by a firstdistance, a hydrogel with the first pattern whose upper surface, of twosurfaces perpendicular to the Z direction, on the side farther apartfrom the bottom surface adheres to the adhesion surface is formed. Afterthat, by displacing the Z stage with the adhesion surface, to which theupper surface of the first pattern adheres, in a direction in which theZ stage moves away from the bottom surface, a lower surface, of the twosurfaces perpendicular to the Z direction, of the first pattern on theside closer to the bottom surface is moved away from the bottom surfaceby a second distance. By performing the second step with the firstpattern being located in the electrolytic solution such that the lowersurface is apart from the bottom surface by the second distance, ahydrogel with the second pattern connecting to the lower surface of thefirst pattern is formed.

In an invention of Claim 6, in the invention of Claim 5, the adhesionsurface is subjected to treatment that makes the adhesion surface havean affinity for water.

In an invention of Claim 7, in the invention of any one of Claims 2 to6, all of the first working electrode groups are included in the secondworking electrode group and the three-dimensionally defined pattern hasa structure in which the first pattern is supported from below by thesecond pattern.

In an invention of Claim 8, in the invention of Claim 5 or 6, all of thesecond working electrode groups are included in the first workingelectrode group and the three-dimensionally defined pattern has astructure in which the second pattern is supported from above, which isthe side where the adhesion surface is located, by the first pattern soas to hang therefrom.

In an invention of Claim 9, in the invention of any one of Claims 1 to8, all of the working electrodes which compose the two-dimensional arrayprotrude from the bottom surface and side faces of each protrudingworking electrode are surrounded with an insulating layer formed so asto protrude from the bottom surface along with the working electrode.

In an invention of Claim 10, in the invention of any one of Claims 1 to9, a part of the bottom surface other than front surfaces of the workingelectrodes is made water-repellent.

In an invention of Claim 11, the invention of any one of Claims 1 to 10further includes a step of forming, on the bottom surface, a hydrogelwith the two-dimensionally defined pattern corresponding to thearrangement of the selected working electrodes by applying thepredetermined voltage to the selected working electrodes, and afterthat, in a state in which the formed hydrogel is in contact with theselected working electrodes, promoting separation of the hydrogel fromthe selected working electrodes by dissolving a contact part of thehydrogel with the selected working electrodes by applying a voltage forhydrogel separation, which is different from the predetermined voltage,to the selected working electrodes.

According to an invention of Claim 12, in a method of producing ahydrogel patterned with cells, which is obtained by distributingpredetermined cells over a hydrogel scaffold, by performing theinvention of any one of Claims 1 to 11 by using, as an electrolyticsolution containing a dissolved substance which causes electrolyticdeposition of the hydrogel, a suspension further containing thepredetermined cells, a hydrogel with the distributed predetermined cellsis formed on the bottom surface of the electrolytic tank.

In an invention of Claim 13, in the invention of Claim 12, thepredetermined cells include one or more types of cells. The invention ofClaim 12 includes a step of performing the invention of Claim 2 byusing, as the electrolytic solution, a suspension containing one type ofcell of the predetermined cells along with a dissolved substance, whichcauses electrolytic deposition of the hydrogel, in any one of the firststep and the second step.

In an invention of Claim 14, the invention of Claim 13 includes a stepof performing the method by using, as the electrolytic solution, asuspension containing the other type of cell, which is different fromthe one type of cell, of the predetermined cells along with a dissolvedsubstance, which causes electrolytic deposition of the hydrogel, in theother of the first step and the second step.

In an invention of Claim 15, in the invention of Claim 12, thepredetermined cells include one or more types of cells, and theinvention of Claim 3 is performed by using, as the electrolyticsolution, a suspension containing one type of cell of the predeterminedcells along with a dissolved substance, which causes electrolyticdeposition of the hydrogel, in any one of the first sub-step and thesecond sub-step.

In an invention of Claim 16, in the invention of Claim 15, the method isperformed by using, as the electrolytic solution, a suspensioncontaining the other type of cell, which is different from the one typeof cell, of the predetermined cells along with a dissolved substance,which causes electrolytic deposition of the hydrogel, in the other ofthe first sub-step and the second sub-step.

In an invention of Claim 17, the invention of any one of Claims 12 to 16includes, before a step of forming a hydrogel by applying thepredetermined voltage to the selected working electrodes in a state inwhich the suspension is housed in the electrolytic tank, adielectrophoresis step of densely distributing the predetermined cellson the selected working electrodes by dielectrophoresis by applying analternating voltage of a predetermined frequency to the selected workingelectrodes.

In an invention of Claim 18, in the invention of Claim 17, in thedielectrophoresis step, an electrode with an electrode plane facing,parallel to the bottom surface, a region of the bottom surface in whichthe two-dimensional array of the working electrodes is provided is used.

In an invention of Claim 19, in the invention of Claim 17, in thedielectrophoresis step, an electrode which is provided on the bottomsurface so as to be located around each of the two-dimensional array ofthe working electrodes is used.

In an invention of Claim 20, the invention of any one of Claims 12 to 16includes, before a step of forming a hydrogel by applying thepredetermined voltage to the selected working electrodes in a state inwhich the suspension is housed in the electrolytic tank, anelectrophoresis step of densely distributing the predetermined cells onthe selected working electrodes by electrophoresis by applying apredetermined direct voltage to the selected working electrodes.

According to an invention of Claim 21, a hydrogel production apparatusthat includes an electrolytic tank with a bottom surface on which atwo-dimensional array of working electrodes is provided and a counterelectrode, which is installed in the electrolytic tank, and can apply apredetermined voltage to one or more selected working electrodes of thetwo-dimensional array of the working electrodes includes a Z stage thathas an adhesion surface facing the bottom surface parallel thereto andis displaceable in a Z direction perpendicular to the bottom surface.

In an invention of Claim 22, in the invention of Claim 21, the adhesionsurface is subjected to treatment that makes the adhesion surface havean affinity for water.

According to an invention of Claim 23, in a hydrogel productionapparatus that includes an electrolytic tank with a bottom surface onwhich a two-dimensional array of working electrodes is provided and acounter electrode, which is installed in the electrolytic tank, and canapply a predetermined voltage to one or more selected working electrodesof the two-dimensional array of the working electrodes, all of theworking electrodes that make up the two-dimensional array protrude fromthe bottom surface and side faces of each protruding working electrodeare surrounded with an insulating layer formed so as to protrude fromthe bottom surface along with the working electrode.

In an invention of Claim 24, in the invention of Claim 21 or 22, all ofthe working electrodes that make up the two-dimensional array protrudefrom the bottom surface and side faces of each protruding workingelectrode are surrounded with an insulating layer formed so as toprotrude from the bottom surface along with the working electrode.

In an invention of Claim 25, in the invention of any one of Claims 21 to24, a part of the bottom surface other than front surfaces of theworking electrodes is made water-repellent.

In an invention of Claim 26, the invention of any one of Claims 21 to 25includes an oscillator that can apply an alternating voltage of apredetermined frequency to the selected working electrodes.

In an invention of Claim 27, the invention of Claim 26 further includesan electrode for dielectrophoresis that is installed or can be installedin the electrolytic tank, and the electrode for dielectrophoresis isinstalled or can be installed in the electrolytic tank so that anelectrode plane thereof faces, parallel to the bottom surface, a regionof the bottom surface in which the two-dimensional array of the workingelectrodes is provided.

In an invention of Claim 28, in the invention of Claim 26, theelectrolytic tank further includes an electrode for dielectrophoresisand the electrode for dielectrophoresis is provided on the bottomsurface so as to be located around each of the two-dimensional array ofthe working electrodes.

According to an invention of Claim 29, in a transducer that includes anelectrolytic tank, which can house an electrolytic solution and ahydrogel which is electrolytically deposited in the electrolyticsolution, mounted on an LSI chip and is used for electrochemicalproduction of a patterned hydrogel, in a shaping area defined on abottom surface of the electrolytic tank, first electrodes which areprovided in the LSI chip in a two-dimensional array arrangement arelocated. The transducer includes a Z stage with an adhesion surface,which can be installed in the electrolytic tank. The Z stage can beinstalled in the electrolytic tank so as be displaceable in a Zdirection perpendicular to the bottom surface, in such a way that theadhesion surface faces the shaping area of the bottom surface parallelto the shaping area.

In an invention of Claim 30, in the invention of Claim 29, the adhesionsurface is subjected to treatment that makes the adhesion surface havean affinity for water.

According to an invention of Claim 31, in a transducer that includes anelectrolytic tank, which can house an electrolytic solution and ahydrogel which is electrolytically deposited in the electrolyticsolution, mounted on an LSI chip and is used for electrochemicalproduction of a patterned hydrogel, in a shaping area defined on abottom surface of the electrolytic tank, first electrodes which areprovided in the LSI chip in a two-dimensional array arrangement arelocated, and all of the first electrodes protrude from the bottomsurface and side faces of each protruding first electrode are surroundedwith an insulating layer formed so as to protrude from the bottomsurface along with the first electrode.

In an invention of Claim 32, in the invention of any one of Claims 29 to31, a part of the shaping area other than front surfaces of the firstelectrodes is made water-repellent.

In an invention of Claim 33, in the invention of any one of Claims 29 to31, in the shaping area, a second electrode which is provided in the LSIchip so as to be located around each of the first electrodes is furtherlocated.

In an invention of Claim 34, in the invention of Claim 33, the secondelectrode is formed over the entire surface of the shaping area withoutcontinuity between the first electrodes and the second electrode.

In an invention of Claim 35, in the invention of Claim 33 or 34, a partof the shaping area other than front surfaces of the first electrodesand a front surface of the second electrode is made water-repellent.

In an invention of Claim 36, the invention of any one of Claims 29 to 32further includes a second electrode that is installed or can beinstalled in the electrolytic tank, and the second electrode isinstalled or can be installed in the electrolytic tank so that anelectrode plane thereof faces the shaping area of the bottom surfaceparallel to the shaping area.

Effects of the Invention

According to the present invention, it is possible to achieve a higherdegree of flexibility in shaping, achieve greater versatility, and forma hydrogel with a two-dimensional pattern or a three-dimensional patternmore efficiently than ever before.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram for explaining the configuration of a hydrogelproduction apparatus of an embodiment.

FIG. 2 is a diagram for explaining the configuration of a transducer ofan embodiment: (a) a plan view; (b) a sectional view; (c) a diagramdepicting the arrangement of cells of an LSI chip; and (d) a diagramdepicting a working electrode provided in each cell.

FIG. 3 is a diagram for explaining electrochemical hydrogel production.

FIG. 4 is a diagram for explaining a method of electrochemicallyproducing a hydrogel (an embodiment).

FIG. 5 is a photograph of a hydrogel actually produced by the method ofelectrochemically producing a hydrogel (an embodiment).

FIG. 6 is a diagram for explaining the method of electrochemicallyproducing a hydrogel (an embodiment).

FIG. 7A is a diagram for explaining a method of forming the workingelectrode protruding from a bottom surface of an electrolytic tank.

FIG. 7B is a diagram for explaining the formation and separation of ahydrogel which are performed when the working electrode depicted in FIG.7A is used.

FIG. 8 is a diagram for explaining the method of electrochemicallyproducing a hydrogel (an embodiment).

FIG. 9 is a diagram for explaining a method of producing a hydrogel witha pattern formed of cells (an embodiment).

FIG. 10 is a diagram for explaining dielectrophoresis which is performedto concentrate cells on the working electrode.

FIG. 11 is a diagram depicting an example of an electrode fordielectrophoresis: (a) an example of an annular electrode; and (b) anexample of a solid electrode.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The principles and embodiments of a technique of electrochemicallyproducing a hydrogel according to the present invention will bedescribed with reference to the drawings.

First, the configuration of a hydrogel production apparatus will bedescribed; then, the configuration of a transducer will be described.

First Embodiment

FIG. 1 is a schematic diagram explaining the configuration of thehydrogel production apparatus. A hydrogel production apparatus 800illustrated as an example includes an electrolytic tank 10, workingelectrodes 20, one counter electrode 30, one reference electrode 40, anda controller 85. The electrolytic tank 10 can house an electrolyticsolution 11 and a hydrogel which is electrolytically deposited in theelectrolytic solution 11. The working electrodes 20, the counterelectrode 30, and the reference electrode 40 are attached in theelectrolytic tank 10 in this example. The working electrodes 20 arearranged on a bottom surface 10 a of the electrolytic tank 10 withpredetermined pitches in the form of a two-dimensional array (example: atwo-dimensional grid-like arrangement; a detailed illustration thereofis omitted in FIG. 1). The controller 85 applies a voltage between thecounter electrode 30 and one or more selected working electrodes 20selected from among the working electrodes 20. The controller 85includes a potentiostat 50 and switches 60. In FIG. 1, a referencenumeral 70 denotes a salt bridge.

The counter electrode 30 and the reference electrode 40 are connected tothe potentiostat 50. Each of the working electrodes 20 is connected tothe potentiostat 50 via a corresponding one of the switches 60. Thepotentiostat 50 functions as a constant-voltage power supply device andis depicted as an equivalent circuit including a variable power source51 and a voltmeter 52 (see FIG. 1). The application of a voltage betweenthe counter electrode 30 and the working electrodes 20 is performed bythe potentiostat 50.

The basic circuit configuration of the potentiostat 50 is the same asthe basic circuit configuration of a potentiostat which is used forcommon electrochemical measurement. That is, a potentiostat which isused for common electrochemical measurement can be used as part of thehydrogel production apparatus without any special change. For thisreason, in the example of FIG. 1, the potentiostat 50 includes anammeter 53 like a potentiostat which is used for common electrochemicalmeasurement.

The controller 85 applies a predetermined voltage between the workingelectrode 20 selected by operation (ON/OFF) performed on the switch 60and the one counter electrode 30. The hydrogel production apparatus 800can further have a configuration which is useful for production of ahydrogel (a detailed illustration thereof is omitted in FIG. 1). Thisuseful configuration will be described later.

Next, the transducer will be described. The transducer includes thehydrogel production apparatus and an integrated circuit. The bottomsurface of the electrolytic tank is the front surface of the integratedcircuit. The configuration of a transducer 900 will be described withreference to FIG. 2.

The transducer 900 has a configuration in which the electrolytic tank 10covers an LSI chip 80. A hole 12 in the shape of a rectangle is formedin the center of the bottom of the electrolytic tank 10. The LSI chip 80is disposed at the lower end of the hole 12 and closes the hole 12.

The LSI chip 80 and the electrolytic tank 10 are fixed on a substrate90. A large number of wiring patterns 91 for connection with an externaldevice (which is not depicted in the drawing) which performs control ofthe LSI chip 80 are formed on the substrate 90. In FIG. 2(b), areference numeral 92 denotes bonding wires that connect the LSI chip 80and the wiring patterns 91.

A shaping area 81 (a diagonally hatched portion in FIG. 2(a)) forproducing a hydrogel with a pattern is set on the upper surface of theLSI chip 80. Specifically, the shaping area 81 is set in the position ofthe hole 12 of the electrolytic tank 10 and faces the electrolyticsolution 11. In the position of the hole 12, the shaping area 81 is partof the bottom surface 10 a of the electrolytic tank 10.

In this example, as depicted in FIG. 2(c), cells 82 are arranged in theshaping area 81 in the form of a two-dimensional grid array. One workingelectrode (a first electrode) 20 is formed in each cell 82 (see FIGS.2(c) and (d)). In this example, 20×20=400 cells 82 are arranged with250-μm pitches in the form of a two-dimensional grid array. That is, theLSI chip 80 has 400 working electrodes 20 arranged in the form of atwo-dimensional grid array.

The LSI chip 80 has a function of applying a voltage between the counterelectrode 30 and each working electrode 20, a switching function, and soforth. The one counter electrode 30 and the one reference electrode 40are installed in the electrolytic solution 11. The reference electrode40 is located immediately above the shaping area 81, and the counterelectrode 30 is located apart from the reference electrode 40 (see FIG.2(b)). The transducer 900 can further have a configuration which isuseful for production of a hydrogel (a detailed illustration thereof isomitted in FIG. 2). This useful configuration will be described later.

Here, the principles of production of a hydrogel by an electrochemicalmethod will be described.

A method of electrochemically producing a hydrogel is classified intothree types.

1) Gelation of dispersoids caused by cross-linking2) Cohesion of charged polymers caused by electrostatic interaction3) Transformation of monomers into polymers by polymerization

FIG. 3 depicts how a hydrogel is produced by a cross-linking reaction,with chitosan being taken up as an example. When a voltage is appliedbetween the counter electrode (an electrode) 30 and the workingelectrode (an electrode) 20 in an aqueous solution in which chitosan andchlorine ions are present, hypochlorous acid (HCIO) is generated by anelectrode reaction (see FIG. 3(b)). Chitosan is oxidized by hypochlorousacid (see FIG. 3(a)). Then, a cross-linking reaction occurs between thechitosan molecules, and a hydrogel 100, which is insoluble in a solvent,is deposited on the working electrode 20.

The example is not limited to chitosan; for example, a hydrogel ofgelatin or sodium alginate can also be produced based on a cross-linkingreaction. Examples of specific conditions of aqueous solutions in caseswhere chitosan is used and gelatin is used are as follows.

-   -   chitosan: 1% (w/v) solution (0.15 M NaCl, adjusted by acetic        acid so as to be pH 5.5)    -   gelatin: 10% (w/v) solution (4 M NaCl, 10 mM acetate buffer        adjusted so as to be pH 5.6)

A method of producing a hydrogel according to the present inventionincludes a process of producing a hydrogel by any one of reactions: 1)cross-linking between dispersoids, 2) electrostatic interaction, and 3)polymerization, which are started by an electrode reaction. The methodof producing a hydrogel uses electrolytic deposition of a hydrogel bywhich a hydrogel is generated by a reaction induced, in an electrolyticsolution, by an electrode product electrochemically generated byelectrodes installed in the electrolytic solution.

According to an embodiment of the present invention, in the hydrogelproduction apparatus 800 and the transducer 900, an electrolyticsolution containing a precursor (i.e., dispersoids, charged polymers,monomers) of a hydrogel as a dissolved substance is housed in theelectrolytic tank (a housing step) and, by applying a predeterminedvoltage between one or more selected working electrodes of the workingelectrodes arranged in the form of a two-dimensional array and the onecounter electrode 30, a hydrogel with a two-dimensional pattern or athree-dimensional pattern corresponding to the arrangement of theselected working electrodes is produced (a production step).

A method of producing a hydrogel with a three-dimensional patternincludes a first step and a second step.

<First Step>

By applying a first voltage, which is a predetermined voltage, betweenall the working electrodes included in a first working electrode groupand one counter electrode, a hydrogel with a first pattern, which is atwo-dimensional pattern, is produced on the bottom surface of theelectrolytic tank. The first working electrode group is made up of oneor more selected working electrodes of the working electrodes 20.

<Second Step>

By applying a second voltage, which is a predetermined voltage, betweenall the working electrodes included in a second working electrode groupand one counter electrode, a hydrogel with a second pattern, which is atwo-dimensional pattern, is produced on the bottom surface of theelectrolytic tank. Since the second pattern connects to the bottomsurface of the first pattern, a hydrogel with a three-dimensionalpattern as a whole is produced on the bottom surface of the electrolytictank. The second working electrode group is made up of one or moreselected working electrodes of the working electrodes 20. At least oneworking electrode included in the first working electrode group makes upthe second working electrode group.

By performing the second step after the first step, a hydrogel with athree-dimensional pattern with a two-layer structure is produced. In thehydrogel with a three-dimensional pattern with a two-layer structure,the hydrogel with the second pattern directly connects to the bottomsurface (a surface, of the two surfaces perpendicular to a Z direction,closer to the bottom surface of the electrolytic tank) of the hydrogelwith the first pattern.

When all the working electrodes included in the first working electrodegroup are included in the second working electrode group, athree-dimensional pattern having a structure in which the first patternis supported from below by the second pattern can be produced.

FIG. 4 is a schematic diagram explaining a process of producing ahydrogel having a structure in which the first pattern is supported frombelow by the second pattern. When a voltage is applied between thecounter electrode 30 and a working electrode 20 ₂, a hydrogel 101 isproduced on the working electrode 20 ₂ as depicted in FIG. 4(2). Next,when a voltage is applied between the counter electrode 30 and workingelectrodes 20 ₁, 20 ₂, and 20 ₃, a hydrogel 102 is produced on theworking electrodes 20 ₁, 20 ₂, and 20 ₃ as depicted in FIG. 4(3). As aresult, a hydrogel 103 with a three-dimensional pattern having astructure in which the hydrogel 101 with the first pattern is supportedfrom below by the hydrogel 102 with the second pattern is produced.

FIG. 5 is a photograph of the actually produced hydrogel with athree-dimensional pattern. This hydrogel was produced by applying avoltage between the counter electrode 30 and the selected workingelectrodes 20 in a chitosan 1% (w/v) aqueous solution with theabove-described conditions by using the transducer 900 depicted in FIG.2. Two voltage application modes (V1 mode, V2 mode) were started at thesame time.

V1 mode: 0 V, 20 s→0.95 V, 10 sV2 mode: 0.95 V, 30 s

Specifically, a voltage in V1 mode was applied between the counterelectrode 30 and the working electrodes 20 of the diagonally hatchedcells 82 (see FIG. 2(c)) and a voltage in V2 mode was applied betweenthe counter electrode 30 and the working electrodes 20 of the cells 82with a dot pattern (see FIG. 2(c)). In other words, a voltage of 0.95 Vwas applied only between the counter electrode 30 and the workingelectrodes 20 of the cells 82 with a dot pattern for first 20 secondsand, for subsequent 10 seconds, a voltage of 0.95 V was applied betweenthe counter electrode 30 and the working electrodes 20 of the diagonallyhatched cells 82 and between the counter electrode 30 and the cells 82with a dot pattern.

It is understood from FIG. 5 that a hydrogel with a three-dimensionalpattern was produced in accordance with the arrangement of the selectedworking electrodes 20 and the voltage application mode (V1 mode, V2mode). In this experiment, the constituent materials of the workingelectrodes 20, the counter electrode 30, and the reference electrode 40of the transducer 900 are as follows.

The working electrodes 20: AuThe counter electrode 30: PtThe reference electrode 40: Ag/AgCl

When a hydrogel with a two-dimensional pattern is produced, the secondstep is unnecessary because a hydrogel with a two-dimensional pattern isproduced only by the first step.

An embodiment is also admissible in which a step of discharging theelectrolytic solution used in the first step from the electrolytic tank10 and housing an electrolytic solution which is used in the second stepin the electrolytic tank 10 is executed between the first step and thesecond step. In this case, preferably, the electrolytic solution whichis used in the second step is different from the electrolytic solutionused in the first step (at least one of the solvent and the dissolvedsubstance of the electrolytic solution which is used in the second stepis different from that of the electrolytic solution which is used in thefirst step). According to such an embodiment, it is possible to producea hydrogel with a three-dimensional pattern with an alginate hydrogel ona chitosan hydrogel, for example.

Second Embodiment

A hydrogel with a three-dimensional pattern with a layered structuresuch as the above-mentioned two-layer structure can be satisfactorilyobtained by using a Z stage. The Z stage is a device that has anadhesion surface parallel to the bottom surface of the electrolytic tankand can move the adhesion surface in the Z direction perpendicular tothe bottom surface of the electrolytic tank.

A second embodiment of the hydrogel production apparatus includes the Zstage in addition to the configuration depicted in FIG. 1. A secondembodiment of the transducer includes the Z stage in addition to theconfiguration depicted in FIG. 2. The Z stage included in the hydrogelproduction apparatus has a configuration in which the Z stage isindependent of the hydrogel production apparatus or a configuration inwhich the Z stage is incorporated into the hydrogel productionapparatus. In the hydrogel production apparatus, the adhesion surface ofthe Z stage is disposed parallel to the bottom surface 10 a of theelectrolytic tank 10. Likewise, the Z stage included in the transducerhas a configuration in which the Z stage is independent of thetransducer or a configuration in which the Z stage is incorporated intothe transducer. In the transducer, the adhesion surface of the Z stageis disposed parallel to the shaping area 81.

The use of the Z stage makes it possible to produce a hydrogel with adesired three-dimensional pattern satisfactorily by the followingprocedure.

a) In the first step, in a state in which the adhesion surface of the Zstage is apart from the bottom surface of the electrolytic tank by afirst distance in the electrolytic solution, a hydrogel with the firstpattern, which is a two-dimensional pattern, is produced on the bottomsurface of the electrolytic tank. The upper surface (a surface, of thetwo surfaces perpendicular to the Z direction, farther apart from thebottom surface) of the produced hydrogel adheres to the adhesion surfaceof the Z stage.

b) After the first step, the adhesion surface of the Z stage moves, withthe hydrogel adhering to the adhesion surface of the Z stage, to aposition apart from the bottom surface of the electrolytic tank by apredetermined distance, which is longer than the first distance, in theelectrolytic solution. The lower surface (a surface, of the two surfacesperpendicular to the Z direction, closer to the bottom surface) of thehydrogel is separated from the bottom surface of the electrolytic tankand apart from the bottom surface of the electrolytic tank by a seconddistance (a separation step).

c) After the separation step, in the second step, in a state in whichthe lower surface of the hydrogel with the first pattern is apart fromthe bottom surface of the electrolytic tank by the second distance, ahydrogel with the second pattern, which is a two-dimensional pattern, isproduced on the bottom surface of the electrolytic tank. Since thesecond pattern connects to the bottom surface of the first pattern, ahydrogel with a three-dimensional pattern as a whole is produced on thebottom surface of the electrolytic tank.

A part, which is immersed in the electrolytic solution, of the Z stageis preferably made of glass, for example, from the viewpoint ofpreventing deterioration caused by an electrochemical reaction. Althoughglass is a material having an affinity for water, it is preferable toperform additional treatment on the adhesion surface of the Z stage toenhance an affinity for water. Examples of this treatment include theapplication of polyethylene glycol, for instance.

FIG. 6 is a schematic diagram depicting a process of producing thehydrogel depicted in FIG. 4 by using the Z stage.

An adhesion surface 110 a of a Z stage 110 is apart from the bottomsurface 10 a of the electrolytic tank by a first distance Z in theelectrolytic solution. By applying a voltage between the counterelectrode 30 and the working electrode 20 ₂ in this state, the hydrogel101 with the first pattern is formed on the working electrode 20 ₂. Asdepicted in FIG. 6(2), the upper surface of the hydrogel 101 adheres tothe adhesion surface 110 a. The separation step in which the hydrogel101 is separated from the working electrode 20 ₂ (see FIG. 6(3)) will bedescribed later in detail.

As depicted in FIG. 6(4), the adhesion surface 110 a of the Z stage 110moves to a position apart from the bottom surface 10 a of theelectrolytic tank by a predetermined distance, which is longer than thefirst distance Z₁, in the electrolytic solution with the hydrogel 101adhering to the adhesion surface 110 a of the Z stage 110. A lowersurface 101 a of the hydrogel 101 is apart from the bottom surface 10 aof the electrolytic tank by a second distance Z₂. By applying a voltagebetween the counter electrode 30 and the working electrodes 20 ₁ to 20 ₃in this state, the hydrogel 102 with the second pattern is produced onthe working electrodes 20, to 20 ₃ as depicted in FIG. 6(5). Thehydrogel 103 with a three-dimensional pattern, which is made up of thehydrogel 101 and the hydrogel 102, is satisfactorily produced.

When all the working electrodes included in the second working electrodegroup are included in the first working electrode group in the processof producing a hydrogel by using the Z stage, a three-dimensionalpattern having a structure in which the first pattern has the secondpattern, which is smaller than the first pattern, hanging therefrom (inother words, when the bottom surface 10 a of the electrolytic tank 10 orthe shaping area 81 is viewed squarely, a structure in which: a) thewhole of the second pattern is included in the first pattern; b) thearea of the second pattern is smaller than the area of the firstpattern; and c) the first pattern is on the second pattern) issatisfactorily produced. That is, a hydrogel with an invertedpyramid-shaped pattern is satisfactorily produced.

The following is an additional explanation of the hydrogel separationstep (see FIG. 6(3)).

The separation step is performed in a state in which the hydrogel 101with the first pattern is in contact with the working electrode 20 ₂. Inthe separation step, a hydrogel separation voltage, which is differentfrom the voltage used in the production of the hydrogel 101, is appliedbetween the counter electrode 30 and the working electrode 20 ₂. As aresult of the application of the hydrogel separation voltage, a part ofthe hydrogel 101 in which the hydrogel 101 is in contact with theworking electrode 20 ₂ is dissolved, and the hydrogel 101 is separatedfrom the working electrode 20 ₂. The separation step allows the hydrogel101 to move with the Z stage 110 while adhering to the adhesion surface110 a of the Z stage 110 (see FIG. 6(4)).

The separation step using the hydrogel separation voltage is useful whena sodium alginate solution, for instance, is used for production of ahydrogel. The reason is as follows: in the case of a sodium alginatesolution, when a voltage which causes an oxidation reaction is appliedbetween the counter electrode and the working electrode, an alginatehydrogel is deposited and, when a voltage which causes a reductionreaction is applied between the counter electrode and the workingelectrode, the alginate hydrogel is dissolved.

However, the separation step using the hydrogel separation voltage isnot an indispensable step. Easy separation of the hydrogel from theworking electrode is made possible by, for example, reducing the contactarea between the hydrogel and the bottom surface 10 a of theelectrolytic tank. In this mode example, all the working electrodesarranged in the form of a two-dimensional array protrude from the bottomsurface of the electrolytic tank. The side faces of the workingelectrodes are surrounded with insulators protruding from the bottomsurface of the electrolytic tank.

FIGS. 7A(1)-(3) are schematic diagrams depicting an example of a methodof producing the working electrodes and the insulators which protrudefrom the bottom surface of the electrolytic tank. As depicted in FIG.7A(2), insulators 25 which form micro wells are produced on the workingelectrodes 20 by photolithography. Then, electrodes are produced byfilling the insides of the micro wells with plating. As a result, asdepicted in FIG. 7A(3), working electrodes 20 a which protrude from thebottom surface 10 a of the electrolytic tank and have the side facessurrounded with the insulators 25 are produced.

A hydrogel 104 produced by applying a voltage between the counterelectrode 30 and the working electrodes 20 a has a shape which does notmake contact with the bottom surface 10 a as depicted in FIG. 7B(2).Thus, the hydrogel 104 is easily separated from the working electrodes20 a at the time of movement of the Z stage 10 depicted in FIG. 7B(3).

Alternatively, easy separation of the hydrogel from the workingelectrode is made possible by, for example, the bottom surface 10 a,which is rendered water-repellent, of the electrolytic tank. In thismode example, the bottom surface of the electrolytic tank other than thefront surface of the working electrode is rendered water-repellent.Rendering the bottom surface water-repellent is, for example, coatingthe bottom surface with a thin film of fluorocarbon resin such aspolytetrafluoroethylene (PTFE).

The separation step using the hydrogel separation voltage can also beapplied to other embodiments.

Third Embodiment

Next, a method of producing a hydrogel with a three-dimensional patternwith a layered structure, in which different types of hydrogel arepresent in the same layer, will be described.

At least one of the above-described first step and second step includesa first sub-step and a second sub-step.

When the first step includes the first sub-step and the second sub-step,the first working electrode group includes a first sub-group and asecond sub-group, the first voltage includes a first sub-voltage and asecond sub-voltage, the first pattern includes a first sub-pattern and asecond sub-pattern, and the second sub-group is made up of only theworking electrode which does not belong to the first sub-group (in otherwords, the first sub-group and the second sub-group do not have a commonworking electrode).

When the second step includes the first sub-step and the secondsub-step, the second working electrode group includes a first sub-groupand a second sub-group, the second voltage includes a first sub-voltageand a second sub-voltage, the second pattern includes a firstsub-pattern and a second sub-pattern, and the second sub-group is madeup of only the working electrode which does not belong to the firstsub-group (in other words, the first sub-group and the second sub-groupdo not have a common working electrode).

In this case, by applying the first sub-voltage between each workingelectrode included in the first sub-group and one counter electrode, ahydrogel with the first sub-pattern, which is a two-dimensional pattern,is produced, and, by applying the second sub-voltage between eachworking electrode included in the second sub-group and the one counterelectrode, a hydrogel with the second sub-pattern, which is atwo-dimensional pattern, is produced. Thus, different types of hydrogelare present in the same layer.

In the first sub-step and the second sub-step, for example, an electrodereaction in the first sub-step is an electrode reaction of oxidation andan electrode reaction in the second sub-step is an electrode reaction ofreduction.

In the case of sodium alginate, a hydrogel is produced by an oxidationreaction. In the case of chitosan, in accordance with pH adjustment, ahydrogel is produced by an oxidation reaction under certain pHconditions and a hydrogel is produced by a reduction reaction underother pH conditions. When pH is set at conditions under which a chitosanhydrogel is produced by a reduction reaction, two types of hydrogel (achitosan hydrogel and an alginate hydrogel) are produced in the firstsub-step and the second sub-step.

FIG. 8 depicts an example in which each of the first step and the secondstep includes the first sub-step and the second sub-step. Specifically,FIG. 8 is a schematic diagram explaining production of a hydrogel inwhich, in a hydrogel with a multi-layer structure, each layer is formedof two types of hydrogel and two types of hydrogel are alternatelylocated in the thickness direction (the Z direction) of the hydrogel. InFIG. 8, a reference numeral 105 denotes an alginate hydrogel and areference numeral 106 denotes a chitosan hydrogel.

FIG. 8(2) depicts the first sub-step in the first step. The electrolytictank 10 is filled with a sodium alginate solution. By applying the firstsub-voltage (V_(s1)) between the first sub-group (the working electrode20 ₂) and the counter electrode 30, the alginate hydrogel 105 with thefirst sub-pattern is produced (the result of an oxidation reaction).

After the sodium alginate solution is removed from the electrolytic tank10, the electrolytic tank 10 is filled with a chitosan solution.

FIG. 8(3) depicts the second sub-step in the first step. Theelectrolytic tank 10 is filled with the chitosan solution. By applyingthe second sub-voltage (V_(s2)) between the second sub-group (theworking electrodes 20, and 20 ₃) and the counter electrode 30, thechitosan hydrogel 106 with the second sub-pattern is produced (theresult of a reduction reaction).

Furthermore, though not depicted in the drawing, the first sub-step inthe second step is executed. The electrolytic tank 10 is filled with thechitosan solution. By applying the first sub-voltage (V_(s2)) betweenthe first sub-group (the working electrode 20 ₂) and the counterelectrode 30, the chitosan hydrogel 106 with the first sub-pattern isproduced (the result of a reduction reaction). That is, the chitosanhydrogel 106 is produced under the alginate hydrogel 105 depicted inFIG. 8(3).

After the chitosan solution is removed from the electrolytic tank 10,the electrolytic tank 10 is filled with the sodium alginate solution.

Though not depicted in the drawing, the second sub-step in the secondstep is executed. The electrolytic tank 10 is filled with the sodiumalginate solution. By applying the second sub-voltage (V_(s1)) betweenthe second sub-group (the working electrodes 20 ₁ and 20 ₃) and thecounter electrode 30, the alginate hydrogel 105 with the secondsub-pattern is produced (the result of an oxidation reaction). That is,the alginate hydrogel 105 is produced under the chitosan hydrogel 106depicted in FIG. 8(3).

By repeating the above-described procedure, a hydrogel 107 with amulti-layer structure (a four-layer structure in the example depicted inFIG. 8(4)) is produced.

In addition, an embodiment in which the above-described sub-steps areperformed is permitted to have a modification including a sub-patternremoval step in which a selected hydrogel is removed. In the sub-patternremoval step, as one example, a hydrogel which is dissolved bypredetermined treatment is produced in the second sub-step and, byperforming the predetermined treatment after the first sub-step and thesecond sub-step, the hydrogel with the second sub-pattern is dissolved.

Specifically, for example, degradation of the alginate hydrogel by theaddition of an alginate degrading enzyme is used. After the alginatehydrogel with the second sub-pattern is produced, the alginate degradingenzyme is diffused in the electrolytic solution. As a result, the secondsub-pattern is selectively removed. The example is not limited to anenzyme; cross-linking scission by light irradiation, dissolution of gelby control of any one of pH and the temperature or both, and the likecan also be used in the sub-pattern removal step.

When a hydrogel with a two-dimensional pattern is produced, as describedearlier, only the first step is executed. In this case, a mode in whichthe first step includes the first sub-step and the second sub-step isalso included in the embodiments of the present invention.

Fourth Embodiment

Next, a method of producing a hydrogel with a pattern formed of cellswill be described. In this example, predetermined cells are distributedover a hydrogel which is a scaffold.

In the method of producing a hydrogel with a pattern formed of cells, asuspension containing a precursor of a hydrogel and predetermined cellsis used as the electrolytic solution. By using this suspension, it ispossible to produce a hydrogel, in which the predetermined cells aredispersed, on the bottom surface of the electrolytic tank.

The above-described method of producing a hydrogel is divided into thefollowing four types.

a) A method including only the first stepb) A method including only the first step, which includes the firstsub-step and the second sub-stepc) A method including the first step and the second stepd) A method including the first step and the second step, at least oneof which includes the first sub-step and the second sub-step

In the case of a), a suspension containing a precursor of a hydrogel andpredetermined cells is used as the electrolytic solution which is usedin the first step.

In the case of b), in at least one of the first sub-step and the secondsub-step, a suspension containing a precursor of a hydrogel and one typeof cell, for example, is used as the electrolytic solution. Theelectrolytic solution which is used in the first sub-step and theelectrolytic solution which is used in the second sub-step may bedifferent from each other (at least one of the solvent and the dissolvedsubstance of the electrolytic solution which is used in the firstsub-step and that of the electrolytic solution which is used in thesecond sub-step are different from each other). For instance, asuspension containing a precursor X of a hydrogel and one type of cellY1 is used as the electrolytic solution which is used in the firstsub-step and a suspension containing the precursor X of a hydrogel andone type of cell Y2 (Y1≠Y2) is used as the electrolytic solution whichis used in the second sub-step.

In the case of c), in at least one of the first step and the secondstep, a suspension containing a precursor of a hydrogel and one type ofcell, for example, is used as the electrolytic solution. By using thissuspension, any one of a hydrogel with the cells dispersed therein andwith the first pattern and a hydrogel with the cells dispersed thereinand with the second pattern or both is produced.

In the case of c), the electrolytic solution which is used in the firststep and the electrolytic solution which is used in the second step maybe different from each other (at least one of the solvent and thedissolved substance of the electrolytic solution which is used in thefirst step and that of the electrolytic solution which is used in thesecond step are different from each other). For instance, a suspensioncontaining a precursor X of a hydrogel and one type of cell Y1 is usedas the electrolytic solution which is used in the first step and asuspension containing the precursor X of a hydrogel and one type of cellY2 (Y1≠Y2) is used as the electrolytic solution which is used in thesecond step. As a result, a hydrogel with the cells Y1 dispersed thereinand with the first pattern and a hydrogel with the cells Y2 dispersedtherein and with the second pattern are produced.

As in the case of c), in the case of d), in at least one of the firstsub-step and the second sub-step, a suspension containing a hydrogelprecursor and one type of cell, for example, is used as the electrolyticsolution.

Furthermore, as in the case of c), in the case of d), the electrolyticsolution which is used in the first sub-step and the electrolyticsolution which is used in the second sub-step may be different from eachother (at least one of the solvent and the dissolved substance of theelectrolytic solution which is used in the first sub-step and that ofthe electrolytic solution which is used in the second sub-step aredifferent from each other).

FIG. 9 is a schematic diagram depicting a process of production of amulti-layer structure formed of an alginate hydrogel in which cells Aare dispersed and a chitosan hydrogel in which cells B are dispersed.

The electrolytic tank 10 is filled with a suspension containing sodiumalginate and the cells A (see FIG. 9(1)).

FIG. 9(2) depicts the first sub-step in the first step. By applying thefirst sub-voltage (V_(s1)) between the first sub-group (the workingelectrode 20 ₂) and the counter electrode 30, an alginate hydrogel 105 awith the first sub-pattern and with the cells A dispersed therein isproduced (the result of an oxidation reaction).

After the suspension containing sodium alginate and the cells A isremoved from the electrolytic tank 10 (see FIG. 9(3)), the electrolytictank is filled with a suspension containing chitosan and the cells B(see FIG. 9(4)).

FIG. 9(5) depicts the second sub-step in the first step. Theelectrolytic tank 10 is filled with the suspension containing chitosanand the cells B. By applying the second sub-voltage (V_(s2)) between thesecond to sub-group (the working electrodes 20, and 20 ₃) and thecounter electrode 30, a chitosan hydrogel 106 a with the secondsub-pattern and with the cells B dispersed therein is produced (theresult of a reduction reaction).

Furthermore, though not depicted in the drawing, the first sub-step inthe second step is executed. The electrolytic tank 10 is filled with thesuspension containing chitosan and the cells B. By applying the firstsub-voltage (V_(s2)) between the first sub-group (the working electrode20 ₂) and the counter electrode 30, the chitosan hydrogel 106 a with thefirst sub-pattern and with the cells B dispersed therein is produced(the result of a reduction reaction). That is, the chitosan hydrogel 106a is produced under the alginate hydrogel 105 a depicted in FIG. 9(6).

After the suspension containing chitosan and the cells B is removed fromthe electrolytic tank 10, the electrolytic tank 10 is filled with thesuspension containing sodium alginate and the cells A.

Though not depicted in the drawing, the second sub-step in the secondstep is executed. The electrolytic tank 10 is filled with the suspensioncontaining sodium alginate and the cells A. By applying the secondsub-voltage (V_(s1)) between the second sub-group (the workingelectrodes 20 ₁ and 20 ₃) and the counter electrode 30, the alginatehydrogel 105 a with the second sub-pattern and with the cells Adispersed therein is produced (the result of an oxidation reaction).That is, the alginate hydrogel 105 a is produced under the chitosanhydrogel 106 a depicted in FIG. 9(6).

By repeating the above-described procedure, a hydrogel 107 a with amulti-layer structure (a three-layer structure in the example depictedin FIG. 9(7)) is produced.

As described above, predetermined cells are dispersed in a hydrogel in astructural arrangement with a three-dimensional design. Thus, thepresent invention is useful for producing a desired cell culturestructure.

An embodiment that produces a hydrogel in which predetermined cells aredispersed is permitted to have a modification including theabove-described sub-pattern removal step. The above-describedsub-pattern removal step is performed at the appropriate time in theprocess of producing a hydrogel in which predetermined cells aredispersed.

The type of cell which is used in the present invention is not limitedto a particular type. For example, cells of any mammal, inducedpluripotent stem (iPS) cells, embryonic stem (ES) cells, mesenchymalstem cells, hepatocytes, vascular endothelial cells, fibroblasts, andthe like are used. For instance, in the case of vascular cells, bydisposing vascular cells in a predetermined area of a hydrogel, it ispossible to produce a blood vessel having a predetermined shape.

When a hydrogel with a pattern formed of cells is produced, it ispreferable to use the cells efficiently. For this reason, for instance,it is preferable to perform a dielectrophoresis step in a state in whicha suspension is housed in the electrolytic tank. The dielectrophoresisstep is performed before a step of producing a hydrogel by applying apredetermined voltage between the selected working electrode and thecounter electrode. In the dielectrophoresis step, an alternating voltageof a predetermined frequency is applied between the selected workingelectrode and the counter electrode and cells are densely distributed onthe selected working electrode by dielectrophoresis. The directivity ofphoresis (an affinity of cells to the density of electrical flux lines)is controlled by a frequency. The working electrode and the counterelectrode in the dielectrophoresis step serve as a positive electrodeand a negative electrode which alternate by an alternating voltage.

FIG. 10 is a schematic diagram depicting how dielectrophoresis of cellsis performed. In this example, an electrode 120 with an electrode planeparallel to the bottom surface 10 a of the electrolytic tank 10 is used.The electrode plane of the electrode 120 faces a region in which thetwo-dimensional array of the working electrodes 20 is formed. In FIG.10, a reference numeral 130 denotes an oscillator that applies analternating voltage of a predetermined frequency between the workingelectrode 20 and the electrode 120. Moreover, a circle represents a celland a dashed line represents an electrical flux line.

In the dielectrophoresis step, an electrode formed on the bottom surface10 a may be used in place of the electrode 120.

In a first example, for each working electrode 20, one electrode 121 fordielectrophoresis, which surrounds the working electrode 20, is formedon the bottom surface 10 a (see FIG. 11(a)). Each electrode 121 has aring-like shape. The electrode 121 and the working electrode 20 are notin contact with each other.

In a second example, one plate electrode 122 for dielectrophoresis isformed on the bottom surface 10 a (see FIG. 11(b)). Holes correspondingto the positions of the working electrodes 20 are formed in the plateelectrode 122. When the bottom surface 10 a is viewed squarely, oneworking electrode 20 is located in one hole and the electrode 122 andthe working electrode 20 are not in contact with each other.

When the dielectrophoresis step is performed, the hydrogel productionapparatus 800 and the transducer 900 include the oscillator 130 inaddition to the potentiostat 50. When the dielectrophoresis step isperformed, the electrode 120 for dielectrophoresis, the electrode 121for dielectrophoresis, or the electrode 122 for dielectrophoresis is aconstituent element of the hydrogel production apparatus 800 or thetransducer 900 or is a part which can be attached to the hydrogelproduction apparatus 800 or the transducer 900. When the Z stage 110 isnot used in the production of a hydrogel, the electrode 120 does notinterfere with the Z stage 110. Thus, in this case, the electrode 120can be used as the counter electrode and the counter electrode 30 isunnecessary.

In the case of the transducer, as in the case of the working electrode20 as the first electrode, the electrode 121 or the electrode 122 as asecond electrode is formed in the shaping area 81 of the LSI chip 80. Itis preferable to render a part of the shaping area 81 other than thefront surface of the electrode water-repellent.

Since cells are negatively charged in general, in place ofdielectrophoresis, electrophoresis can also be used to concentrate thecells densely on the selected working electrode. Electrophoresis isperformed by applying a predetermined direct voltage between theselected working electrode and the counter electrode. In anelectrophoresis step which is performed on the negatively charged cells,the working electrode serves as to a positive electrode and the counterelectrode serves as a negative electrode.

According to the present invention, it is possible to achieve a higherdegree of flexibility in shaping, achieve greater versatility, andproduce a hydrogel with a two-dimensional pattern or a three-dimensionalpattern more efficiently than ever before. This makes it possible toproduce a hydrogel of any shape accurately, easily, and quickly.

The present invention will be described as follows from a differentperspective. It is to be noted that the following description is notcontradictory to the disclosure described in the above-mentioned “MEANSTO SOLVE THE PROBLEMS” and it is possible to cross-reference thefollowing description and the above-mentioned “MEANS TO SOLVE THEPROBLEMS” with each other.

Item 1

A method of electrochemically producing a hydrogel by using electrolyticdeposition, the method including:

a step of housing an electrolytic solution containing a precursor of thehydrogel in an electrolytic tank, the electrolytic tank having a counterelectrode therein and having a bottom surface with working electrodesarranged thereon; and

a first step of producing the hydrogel with a first two-dimensionalpattern on the bottom surface by applying a first voltage between thecounter electrode and all the working electrodes included in a firstworking electrode group, the first working electrode group consisting ofone or more ones selected from among the working electrodes.

Item 2

The electrochemical production method described in Item 1, furtherincluding a second step that is executed after the first step,

wherein the second step includes producing on the bottom surface, byapplying a second voltage between the counter electrode and all theworking electrodes included in a second working electrode group, thehydrogel with a three-dimensional pattern in which a secondtwo-dimensional pattern connects to a bottom surface of the firsttwo-dimensional pattern,

wherein the second working electrode group consists of one or more onesselected from among the working electrodes, and

wherein one or more ones included in the first working electrode groupare included in the second working electrode group.

Item 3

The electrochemical production method described in Item 2, including,between the first step and the second step, a step of discharging theelectrolytic solution used in the first step from the electrolytic tankand housing, in the electrolytic tank, the electrolytic solution whichis to be used in the second step,

wherein the electrolytic solution used in the second step is differentfrom the electrolytic solution used in the first step.

Item 4

The electrochemical production method described in Item 2 or 3, whereineither or both of the first step and the second step include a firstsub-step and a second sub-step,

wherein, when the first step includes the first sub-step and the secondsub-step, the first working electrode group includes a first sub-groupand a second sub-group, the first voltage includes a first sub-voltageand a second sub-voltage, the first pattern includes a first sub-patternand a second sub-pattern, and the first sub-group and the secondsub-group do not have a common working electrode,

wherein, when the second step includes the first sub-step and the secondsub-step, the second working electrode group includes a first sub-groupand a second sub-group, the second voltage includes a first sub-voltageand a second sub-voltage, the second pattern includes a firstsub-pattern and a second sub-pattern, and the first sub-group and thesecond sub-group do not have a common working electrode,

wherein, by applying the first sub-voltage between the counter electrodeand all the working electrodes included in the first sub-group, thehydrogel with the first sub-pattern is produced on the bottom surface,and

wherein, by applying the second sub-voltage between the counterelectrode and all the working electrodes included in the secondsub-group, the hydrogel with the second sub-pattern is produced on thebottom surface.

Item 5

The electrochemical production method described in Item 4, including,between the first sub-step and the second sub-step, a step ofdischarging the electrolytic solution used in the first sub-step fromthe electrolytic tank and housing, in the electrolytic tank, theelectrolytic solution which is to be used in the second sub-step,

wherein the electrolytic solution used in the second sub-step isdifferent from the electrolytic solution used in the first sub-step.

Item 6

The electrochemical production method described in Item 4 or 5, furtherincluding a sub-pattern removal step that is executed after the firstsub-step and the second sub-step,

wherein, in the first sub-step or the second sub-step, the hydrogelwhich is dissolved by predetermined treatment is produced, and

wherein, in the sub-pattern removal step, the hydrogel with the firstsub-pattern or the second sub-pattern is dissolved by performing thepredetermined treatment.

Item 7

The electrochemical production method described in any one of Items 2 to6, further including a separation step that is performed after the firststep and before the second step,

wherein the first step and the second step are performed by using a Zstage that has an adhesion surface parallel to the bottom surface of theelectrolytic tank and moves the adhesion surface in a directionperpendicular to the bottom surface of the electrolytic tank,

wherein in the first step, the hydrogel with the first two-dimensionalpattern is produced between the adhesion surface, which is located apartfrom the bottom surface of the electrolytic tank by a first distance inthe electrolytic solution, and the bottom surface of the electrolytictank and is in contact with the adhesion surface,

wherein in the separation step, in a process whereby the adhesionsurface is moved in the electrolytic solution by the Z stage to aposition apart from the bottom surface of the electrolytic tank by apredetermined distance, which is greater than the first distance, thehydrogel with the first two-dimensional pattern is separated from thebottom surface of the electrolytic tank while adhering to the adhesionsurface, and

wherein in the second step, the hydrogel with the three-dimensionalpattern is produced between the adhesion surface and the bottom surfaceof the electrolytic tank and the second two-dimensional pattern is incontact with a bottom surface of the first two-dimensional pattern.

Item 8

The electrochemical production method described in Item 7, wherein

the adhesion surface has an affinity for water.

Item 9

The electrochemical production method described in any one of Items 2 to8,

wherein the number of working electrodes included in the second workingelectrode group is greater than the number of working electrodesincluded in the first working electrode group, and

wherein all the working electrodes included in the first workingelectrode group are included in the second working electrode group.

Item 10

The electrochemical production method described in Item 7 or 8,

wherein the number of working electrodes included in the first workingelectrode group is greater than the number of working electrodesincluded in the second working electrode group, and

wherein all the working electrodes included in the second workingelectrode group are included in the first working electrode group.

Item 11

The electrochemical production method described in any one of Items 1 to10,

wherein each of the working electrodes protrudes from the bottomsurface, and

wherein side faces of each of the working electrodes are surrounded withinsulators protruding from the bottom surface of the electrolytic tank.

Item 12

The electrochemical production method described in any one of Items 1 to11,

wherein the bottom surface of the electrolytic tank other than frontsurfaces of the working electrodes is water-repellent.

Item 13

The electrochemical production method described in any one of Items 1 to12, further including a step of dissolving, by applying a voltage, whichis different from the first voltage, between the counter electrode andall the working electrodes included in the first working electrode groupin a state in which the hydrogel with the first two-dimensional patternis in contact with a working electrode included in the first workingelectrode group, a part of the hydrogel at which the hydrogel is incontact with the working electrode included in the first workingelectrode group.

Item 14

A method of producing a hydrogel with a pattern formed of cells,including a step of using an electrochemical production method that isthe electrochemical production method described in any one of Items 1 to13 and uses, as an electrolytic solution, a suspension containing aprecursor of the hydrogel and the cells.

Item 15

The production method described in Item 14, further including a step ofconcentrating the cells on the first working electrode group bydielectrophoresis or electrophoresis before the first step or a step ofconcentrating the cells on the second working electrode group bydielectrophoresis or electrophoresis before the second step.

Item 16

A hydrogel production apparatus for producing a hydrogel by usingelectrolytic deposition, including:

an electrolytic tank with a counter electrode set therein, theelectrolytic tank having a bottom surface with working electrodesarranged thereon;

a controller applying a voltage between the counter electrode and one ormore ones selected from among the working electrodes; and

a Z stage having an adhesion surface for the hydrogel's adhesion andmoving the adhesion surface in a direction perpendicular to the bottomsurface of the electrolytic tank while keeping the adhesion surfaceparallel to the bottom surface of the electrolytic tank.

Item 17

The hydrogel production apparatus described in Item 16, wherein theadhesion surface has an affinity for water.

Item 18

The hydrogel production apparatus described in Item 16 or 17,

wherein each of the working electrodes protrudes from the bottomsurface, and

wherein side faces of the working electrodes are surrounded withinsulators protruding from the bottom surface of the electrolytic tank.

Item 18

A hydrogel production apparatus for producing a hydrogel by usingelectrolytic deposition, including:

an electrolytic tank with a counter electrode set therein, theelectrolytic tank having a bottom surface with working electrodesarranged thereon; and

a controller applying a voltage between the counter electrode and one ormore ones selected from among the working electrodes,

each of the working electrodes protruding from the bottom surface, and

side faces of the working electrodes being surrounded with insulatorsprotruding from the bottom surface of the electrolytic tank.

Item 19

The hydrogel production apparatus described in any one of Items 16 to18,

wherein the bottom surface of the electrolytic tank other than frontsurfaces of the working electrodes is water-repellent.

Item 20

The hydrogel production apparatus described in any one of Items 16 to19, further including:

an electrode for phoresis; and

an oscillator that applies an alternating voltage of a predeterminedfrequency between the electrode for phoresis and one or more onesselected from among the working electrodes.

Item 21

A transducer including:

the hydrogel production apparatus described in any one of Items 16 to20; and

an integrated circuit,

the bottom surface of the electrolytic tank being a front surface of theintegrated circuit.

INDUSTRIAL APPLICABILITY

The technique of producing a hydrogel according to the present inventionis useful in, for example, assessment of drugs as an alternative toanimal testing, production of operation practice equipment, productionof medical supplies, and so forth.

1-36. (canceled)
 37. A method of electrochemically producing a hydrogelby using electrolytic deposition of a hydrogel, the method by which ahydrogel is formed by a reaction which is induced in an electrolyticsolution by an electrode product electrochemically generated byelectrodes installed in the electrolytic solution, the methodcomprising: a preparing step of providing an apparatus including anelectrolytic tank and housing an electrolytic solution therein, theelectrolytic tank having a two-dimensional array of working electrodesprovided on a bottom surface thereof and a counter electrode installedtherein, the electrolytic solution containing a dissolved substancewhich causes electrolytic deposition of the hydrogel; a first step offorming a hydrogel with a two-dimensionally defined first pattern on thebottom surface, by applying a first voltage as a predetermined voltageto a first working electrode group as one or more selected workingelectrodes of the two-dimensional array of working electrodes, the firstpattern corresponding to an arrangement of the first working electrodegroup; and a second step of forming a hydrogel with a two-dimensionallydefined second pattern on the bottom surface, by applying a secondvoltage as a predetermined voltage to a second working electrode groupas one or more further selected working electrodes of thetwo-dimensional array of working electrodes, the second patterncorresponding to an arrangement of the second working electrode group,wherein the second step is performed after the first step, and ahydrogel with a three-dimensionally defined pattern including at leasttwo layers is formed, such that one of the two layers having the secondpattern is located under an other of the two layers having the firstpattern on a side thereof closer to the bottom surface.
 38. The methodof electrochemically producing a hydrogel according to claim 37, whereinone of the first step and the second step includes a first sub-step anda second sub-step, a corresponding one of the first working electrodegroup and the second working electrode group includes a first workingelectrode sub-group and a second working electrode sub-group, acorresponding one of the first voltage and the second voltage includes afirst sub-voltage and a second sub-voltage, and a corresponding one ofthe first pattern and the second pattern includes a first sub-patternand a second sub-pattern, the second working electrode sub-groupconsists of only working electrodes which do not belong to the firstworking electrode sub-group, and a hydrogel with the first sub-patternis formed by applying the first sub-voltage to the first workingelectrode sub-group and a hydrogel with the second sub-pattern is formedby applying the second sub-voltage to the second working electrodesub-group.
 39. The method of electrochemically producing a hydrogelaccording to claim 38, wherein in the second sub-step, a hydrogel whichdissolves under a predetermined condition is formed as the hydrogel withthe second sub-pattern, and the method further includes, after at leastboth of the first sub-step and the second sub-step, a sub-patternremoval step of selectively removing the hydrogel with the secondsub-pattern by dissolving the hydrogel with the second sub-pattern byproviding the hydrogel with the second sub-pattern with thepredetermined condition.
 40. The method of electrochemically producing ahydrogel according to claim 37, wherein a Z stage that has an adhesionsurface facing parallel the bottom surface and is displaceable in a Zdirection perpendicular to the bottom surface is further provided, byperforming the first step with the Z stage being located in theelectrolytic solution such that the adhesion surface is apart from thebottom surface by a first distance, the hydrogel with the first patternis formed such that an upper surface thereof adheres to the adhesionsurface, wherein the upper surface is one, on a side farther apart fromthe bottom surface, of two surfaces perpendicular to the Z direction ofthe hydrogel with the first pattern, and after that, by displacing the Zstage, to the adhesion surface of which the upper surface of thehydrogel with the first pattern adheres, in a direction in which the Zstage moves away from the bottom surface, a lower surface of thehydrogel with the first pattern is moved away from the bottom surface bya second distance, wherein the lower surface is an other, on a sidecloser to the bottom surface, of the two surfaces perpendicular to the Zdirection of the hydrogel with the first pattern, and by performing thesecond step with the hydrogel with the first pattern being located inthe electrolytic solution such that the lower surface is apart from thebottom surface by the second distance, the hydrogel with the secondpattern connecting to the lower surface of the hydrogel with the firstpattern is formed.
 41. The method of electrochemically producing ahydrogel according to claim 40, wherein the adhesion surface issubjected to treatment that makes the adhesion surface have an affinityfor water.
 42. The method of electrochemically producing a hydrogelaccording to claim 37, wherein all of the first working electrode groupsare included in the second working electrode group, and the hydrogelwith the three-dimensionally defined pattern has a structure in whichthe hydrogel with the first pattern is supported from below by thehydrogel with the second pattern.
 43. The method of electrochemicallyproducing a hydrogel according to claim 40, wherein all of the secondworking electrode groups are included in the first working electrodegroup, and the hydrogel with the three-dimensionally defined pattern hasa structure in which the hydrogel with the second pattern is supportedfrom above, which is a side where the adhesion surface is located, bythe hydrogel with the first pattern so as to hang therefrom.
 44. Themethod of electrochemically producing a hydrogel according to claim 37,wherein a working electrode included in the two-dimensional array ofworking electrodes protrudes from the bottom surface and a side face ofthe protruding working electrode is surrounded with an insulating layerformed so as to protrude from the bottom surface along with theprotruding working electrode.
 45. The method of electrochemicallyproducing a hydrogel according to claim 37, wherein a part of the bottomsurface other than front surfaces of the working electrodes is madewater-repellent.
 46. The method of electrochemically producing ahydrogel according to claim 37, wherein at least one of the first stepand the second step further comprises a sub-step of facilitatingseparation of the hydrogel having corresponding one of the first patternand the second pattern, after forming thereof, from corresponding one ofthe first working electrode group and the second working electrodegroup, in which a part of the hydrogel that contacts the correspondingworking electrode group is dissolved by applying a hydrogel separatingvoltage to the corresponding working electrode group when the hydrogelis in contact therewith, the hydrogel separating voltage being differentthan corresponding one of the first voltage and the second voltage. 47.A method of electrochemically producing a hydrogel by using electrolyticdeposition of a hydrogel, the method by which a hydrogel is formed by areaction which is induced in an electrolytic solution by an electrodeproduct electrochemically generated by electrodes installed in theelectrolytic solution, the method comprising: a preparing step ofproviding an apparatus including an electrolytic tank and housing anelectrolytic solution therein, the electrolytic tank having atwo-dimensional array of a plurality of working electrodes provided on abottom surface thereof and a counter electrode installed therein, theelectrolytic solution containing a dissolved substance which causeselectrolytic deposition of the hydrogel; a hydrogel formation step offorming, on the bottom surface, by applying a predetermined voltage toone or more selected working electrodes of the two-dimensional array ofthe plurality of working electrodes, a hydrogel with a two-dimensionallydefined pattern corresponding to an arrangement of the selected workingelectrodes; and a separation facilitating step of facilitatingseparation of the hydrogel from the selected working electrodes, afterthe hydrogel formation step, by dissolving a part of the hydrogel thatcontacts the selected working electrodes by applying a voltage forhydrogel separation, which is different from the predetermined voltage,to the selected working electrodes.
 48. A method of producing a hydrogelpatterned with cells, which is obtained by distributing prescribed cellsover a hydrogel scaffold, wherein by performing the method ofelectrochemically producing a hydrogel according to claim 37 by using asuspension as the electrolytic solution containing the dissolvedsubstance which causes electrolytic deposition of the hydrogel, thesuspension further containing the prescribed cells, the hydrogel havingdistributed therein the prescribed cells is formed on the bottom surfaceof the electrolytic tank.
 49. A method of producing a hydrogel patternedwith cells, which is obtained by distributing prescribed cells over ahydrogel scaffold, wherein by performing the method of electrochemicallyproducing a hydrogel according to claim 47 by using a suspension as theelectrolytic solution containing the dissolved substance which causeselectrolytic deposition of the hydrogel, the suspension furthercontaining the prescribed cells, the hydrogel having distributed thereinthe prescribed cells is formed on the bottom surface of the electrolytictank.
 50. A method of producing a hydrogel patterned with cells, whichis obtained by distributing prescribed cells over a hydrogel scaffold,wherein the prescribed cells include one or more types of cells, and themethod includes a step of performing the method of electrochemicallyproducing a hydrogel according to claim 37, wherein one of the firststep and the second step is performed by using a suspension as theelectrolytic solution concerned, the suspension containing one type ofcells included by the one or more types of cells along with thedissolved substance which causes electrolytic deposition of the hydrogelconcerned.
 51. The method of producing a hydrogel patterned with cellsaccording to claim 50, wherein the prescribed cells include at least twotypes of cells, and an other of the first step and the second step isperformed by using an other suspension as the electrolytic solutionconcerned, the other suspension containing an other type of cellsincluded by the at least two types of cells, the other type of cellsbeing different from the one type of cells, along with the dissolvedsubstance which causes electrolytic deposition of the hydrogelconcerned.
 52. A method of producing a hydrogel patterned with cells,which is obtained by distributing prescribed cells over a hydrogelscaffold, wherein the prescribed cells include one or more types ofcells, and the method includes a step of performing the method ofelectrochemically producing a hydrogel according to claim 38, whereinone of the first sub-step and the second sub-step is performed by usinga suspension as the electrolytic solution concerned, the suspensioncontaining one type of cells included by the one or more types of cellsalong with the dissolved substance which causes electrolytic depositionof the hydrogel concerned.
 53. The method of producing a hydrogelpatterned with cells according to claim 52, wherein the prescribed cellsinclude at least two types of cells, and an other of the first sub-stepand the second sub-step is performed by using an other suspension as theelectrolytic solution concerned, the other suspension containing another type of cells included by the at least two types of cells, theother type of cells being different from the one type of cells, alongwith the dissolved substance which causes electrolytic deposition of thehydrogel concerned.
 54. The method of producing a hydrogel patternedwith cells according to claim 48, further comprising, before one of thefirst step and the second step, a dielectrophoresis step of denselydistributing the prescribed cells above corresponding one of the firstworking electrode group and the second working electrode group bydielectrophoresis, by applying an alternating voltage of a predeterminedfrequency to the corresponding one of the first working electrode groupand the second working electrode group.
 55. The method of producing ahydrogel patterned with cells according to claim 49, further comprising,before hydrogel formation step, a dielectrophoresis step of denselydistributing the prescribed cells above the selected working electrodesby dielectrophoresis, by applying an alternating voltage of apredetermined frequency to the selected working electrodes.
 56. Themethod of producing a hydrogel patterned with cells according to claim54, wherein in the dielectrophoresis step, an electrode with anelectrode plane facing, parallel to the bottom surface, a region of thebottom surface in which the two-dimensional array of the workingelectrodes is provided is used.
 57. The method of producing a hydrogelpatterned with cells according to claim 55, wherein in thedielectrophoresis step, an electrode with an electrode plane facing,parallel to the bottom surface, a region of the bottom surface in whichthe two-dimensional array of the working electrodes is provided is used.58. The method of producing a hydrogel patterned with cells according toclaim 54, wherein in the dielectrophoresis step, an electrode which isprovided on the bottom surface so as to be located around each of thetwo-dimensional array of the working electrodes is used.
 59. The methodof producing a hydrogel patterned with cells according to claim 55,wherein in the dielectrophoresis step, an electrode which is provided onthe bottom surface so as to be located around each of thetwo-dimensional array of the working electrodes is used.
 60. The methodof producing a hydrogel patterned with cells according to claim 48,further comprising, before one of the first step and the second step, anelectrophoresis step of densely distributing the prescribed cells abovecorresponding one of the first working electrode group and the secondworking electrode group by electrophoresis, by applying a predetermineddirect voltage to the corresponding one of the first working electrodegroup and the second working electrode group.
 61. The method ofproducing a hydrogel patterned with cells according to claim 49, furthercomprising, before hydrogel formation step, an electrophoresis step ofdensely distributing the prescribed cells above the selected workingelectrodes by electrophoresis, by applying a predetermined directvoltage to the selected working electrodes.
 62. A hydrogel productionapparatus, comprising: an electrolytic tank having a bottom surfacethereof; a two-dimensional array of working electrodes provided on thebottom surface; a counter electrode installed in the electrolytic tank;a controller configured to apply a predetermined voltage to one or moreselected working electrodes of the two-dimensional array of the workingelectrodes; and a Z stage that has an adhesion surface facing parallelthe bottom surface and is displaceable in a Z direction perpendicular tothe bottom surface.
 63. The hydrogel production apparatus according toclaim 62, wherein the adhesion surface is subjected to treatment thatmakes the adhesion surface have an affinity for water.
 64. The hydrogelproduction apparatus according to claim 62, wherein a working electrodeincluded in the two-dimensional array of working electrodes protrudesfrom the bottom surface and a side face of the protruding workingelectrode is surrounded with an insulating layer formed so as toprotrude from the bottom surface along with the protruding workingelectrode.
 65. The hydrogel production apparatus according to claim 62,wherein a part of the bottom surface other than front surfaces of theworking electrodes is made water-repellent.
 66. The hydrogel productionapparatus according to claim 62, further comprising an oscillatorconfigured to apply an alternating voltage of a predetermined frequencyto the selected working electrodes.
 67. The hydrogel productionapparatus according to claim 66, further comprising one of an installedelectrode for dielectrophoresis and an installable electrode fordielectrophoresis, wherein the installed electrode for dielectrophoresisis an electrode installed in the electrolytic tank so that an electrodeplane thereof faces, parallel to the bottom surface, a region of thebottom surface in which the two-dimensional array of the workingelectrodes is provided, and wherein the installable electrode fordielectrophoresis is an electrode adapted to be installed in theelectrolytic tank so that an electrode plane thereof faces, parallel tothe bottom surface, a region of the bottom surface in which thetwo-dimensional array of the working electrodes is provided.
 68. Thehydrogel production apparatus according to claim 66, wherein theelectrolytic tank further includes an electrode for dielectrophoresis,and the electrode for dielectrophoresis is provided on the bottomsurface so as to be located around each of the two-dimensional array ofthe working electrodes.
 69. A transducer configured to be used forelectrochemical production of a patterned hydrogel, comprising: an LSIchip; an electrolytic tank having a bottom surface thereof in which ashaping area is defined, the electrolytic tank being mounted on the LSIchip; a plurality of first electrodes provided in the LSI chip, theplurality of first electrodes being located in the shaping area in anarrangement of a two-dimensional array; and a Z stage having an adhesionsurface thereof, wherein the Z stage is configured to be installed inthe electrolytic tank in such a way that the Z stage is displaceable ina Z direction perpendicular to the bottom surface with the adhesionsurface kept facing parallel the shaping area of the bottom surface. 70.The transducer according to claim 69, wherein the adhesion surface issubjected to treatment that makes the adhesion surface have an affinityfor water.
 71. The transducer according to claim 69, wherein theplurality of first electrodes protrude from the bottom surface and aside face of each of the first electrodes that protrudes is surroundedwith an insulating layer formed so as to protrude from the bottomsurface along with the first electrode.
 72. The transducer according toclaim 69, wherein a part of the shaping area other than front surfacesof the first electrodes is made water-repellent.
 73. The transduceraccording to claim 69, further comprising a second electrode provided inthe LSI chip, the second electrode being located around each of thefirst electrodes in the shaping area.
 74. The transducer according toclaim 73, wherein the second electrode is formed over an entire surfaceof the shaping area without continuity between the first electrodes andthe second electrode.
 75. The transducer according to claim 73, whereina part of the shaping area other than front surfaces of the firstelectrodes and a front surface of the second electrode is madewater-repellent.
 76. The transducer according to claim 69, furthercomprising one of a second electrode and a third electrode, wherein thesecond electrode is installed in the electrolytic tank so that anelectrode plane thereof faces parallel the shaping area of the bottomsurface, and wherein the third electrode is adapted to be installed inthe electrolytic tank so that an electrode plane thereof faces parallelthe shaping area of the bottom surface.